The present invention relates to immunologically active polypeptides with no or reduced toxicity useful for the production of an antipertussis vaccine.
The invention also relates to a method for the preparation of said polypeptides and to an antipertussis vaccine comprising a therapeutically effective amount of at least one of said polypeptides.
Pertussis is a respiratory system disease caused by Bordetella pertussis (B. pertussis), a bacillus the transmission of which occurs during the catarrhal and convulsive phase from a sick person to a healthy predisposed individual through the respiratory system.
A vaccine effective against said disease is particularly desirable since pertussis may cause convulsions, cerebral damages and, sometimes, death, principally in tender age children and in newborn babies lacking maternal antipertussis antibodies obtained passively.
At present, it is employed an antipertussis vaccine comprising virulent bacteria killed with merthiolate and treated at 56xc2x0 C. that, even if it confers a permanent protection, it is not, however, completely satisfactory, either for the presence of undesired side effects or for the numerous problems deriving from the preparation and purification thereof.
This results in the necessity of preparing an antipertussis vaccine lacking of the aforementioned drawbacks.
It is known that B. pertussis has per se, no virulence and that its toxicity is corrolated with the synthesis, during the phase I (virulent), such substances as: hemolysin (Hls), adenylcyclase (Adc), dermonecrotic toxin (Dnc), filamentary hemagglutinin (Fha) and pertussis toxin (PT). The latter, in particular, represents not only the major virulence factor caused by B. pertussis (Weiss A. et al. (1983) Infect, Immun. 42, 333-41; Weiss A. et al. (1984) J. Inf. Dis. 150, 219-222but also one of the major protective antigens against infections caused by said bacterium.
Anti-PT antibodies, in fact, have been found in individuals immunized by the cellular vaccine (Ashworth L. A. E. et al. (1983) Lancet. October 878-881) and a protective immunity has been obtained in mice infected, via aereosol or intracerebrally, using formaldehyde-detoxified PT (Sato Y. et al. (1983) Inf. and Imm. 41, 313). Even if the pertussis toxin represents an essential component in the preparation of new antipertussis vaccines, its use is limited by the numerous drawbacks deriving from its toxicity.
The PT, in fact, induces undesirable pathophysiologic effects such as: lymphocytosis, histamine sensitivity, hypoglycemia, insensitivity to the hyperglycemic effect of epinephrine and activation of the islands of Langerhans.
Furthermore, it has been found that the PT presence in the vaccine now employed is the principal cause of such side effects as: fever, pomphus, neurologic alteration and death which have led, in recent years, to drastically reducing the use of the vaccine with the consequent new outbreak of pertussis cases.
The PT detoxification treatment by means of formaldehyde though allowing to get an immunogenic protein without toxicity (Sato et al. reference reported above), presents some drawbacks deriving from the fact that said protein is not obtainable in pure, reproducible and stable form.
According to that, polypeptides have now been found which are able to overcome the prior art drawbacks and are obtainable in pure form by means of a simple and economically feasible method. One object of the present invention, therefore, consists of immunologically active polypeptides with no or reduced toxicity useful for the preparation of an antipertussis vaccine.
A further object of the present invention consists of a method for the preparation of said polypeptides.
Another object of the present invention is a vaccine comprising a therapeutically effective amount of at least one of said polypeptides.
Further objects of the present invention will become apparent from a reading of the following description and examples.
The pertussis toxin is a protein comprising five different subunits the toxicity of which is due to ADP-ribosylation of proteins which bind GTP involved in the transmission of messages through eukaryotic cells membranes.
Said PT comprises two fractions with different functionality: A comprising the S1 subunit and B comprising S2, S3, S4 and S5 subunits placed in two dimers D1(S2+S4) and D2 (S3+S4) linked to each other by the S5 subunit.
The A fraction represents the enzymatically active and therefore, toxic part of PT, whereas the B fraction is linked to the eukaryotic cells membrane receptors and allows the introduction of the S1 subunit therein.
In the copending Italian patent application No. 19208-A/86, now filed in the United States as U.S. application Ser. No. 07/006,438, filed Jan. 23, 1987, the cloning, sequencing and expression of the genes which code for said subunits have been described and claimed and it has been shown that said genes are grouped in a sole operon.
Furthermore, the ADP-ribosylation activity of the S1 subunit has been determined, by cultivating a microorganism transformed with the hybrid plasmid PTE225, and it has been found that said subunit possesses an enzymatic activity comparable to that of PT.
According to that and to the end of obtaining a protein having the immunologic and protective properties of the pertussis toxin but with no or reduced toxicity, the positions and the fundamental aminoacids for the enzymatic activity of the protein have been identified.
In particular, the following positions and aminoacids have been found:
tyrosine(8), arginine(9), phenylalanine (50), threonine(53), glutamic acid (129), glycine (121), alanine (124), aspartic acid (109), glycine (99), arginine (135), threonine (159) and tyrosine (111).
The substitution of one or more of said amino acids with any aminoacid different from the one which is bound to be changed, allows to obtain a protein with altered toxicity.
According to that, in accordance with the present invention, polypeptides have been synthesized containing S1 subunits of the modified pertussis toxin by means of direct mutagenesis substituting, in one or more positions of the S1 region comprised between the 1-180 amino acids, one aminoacid with another capable of destroying or reducing its enzymatic activity without altering the immunologic properties thereof.
In particular, polypeptides have been synthesized containing the S1 subunit of the pertussis toxin modified by substituting:
the tyrosine in position 8 and arginine in position 9 with aspartic acid and glycine;
the phenylalanine in position 50 and the threonine in position 53 with glutamic acid and isoleucine;
the glutamic acid in position 129 with glycine;
the glycine in position 121 with glutamic acid;
the alanine in position 124 with aspartic acid;
the aspartic acid in position 109 and the alanine in position 124 with glycine and aspartic acid respectively;
the glycine in position 99 with glutamic acid;
the aspartic acid in position 109 with glycine;
the arginine in position 135 with glutamic acid;
the threonine in position 159 with lysine;
the tyrosine in position 111 with glycine and insertion of Asp Thr Gly Gly amminoacids in position 113.
In particular, the polypeptides according to the present invention have been prepared by a method which comprises:
a) modifying by means of direct mutagenesis the gene which codes for the S1 subunit of the pertussis toxin substituting, in one or more sites of the DNA molecule, the bases sequence which codes for a predetermined aminoacid with a bases sequence which codes for the aminoacid of interest;
b) constructing a hybrid plasmid linking a cloning vector with the DNA fragment containing the modified S1;
c) transforming a host microorganism with a hybrid plasmid obtained as reported in b);
d) cultivating in a suitable culture medium, in presence of carbon, nitrogen and mineral salts sources a transformed microorganism and then,
e) recovering the polypeptide containing the modified S1 subunit from the culture medium or from the cells.
According to the present invention and to the end of identifying the S1 aminoacidic region correlated to the enzymatic activity of the protein, the gene which codes for S1 has been treated with restriction enzymes that cut in different sites and the DNA fragments so obtained, lacking of the3xe2x80x2 and/or 5xe2x80x2 of different length terminal sequences, have been cloned in an expression plasmid operating according to one of the generally known techniques The vectors containing the DNA fragments with the deleted sequences have been then employed to transform Escherichia coli (E. coli) cells.
The positive transformants, obtained screening the cells on a selective medium, have been cultivated in a suitable culture medium at temperatures between 30xc2x0 C. and 40xc2x0 C. for a period of from 20 minutes to 5 hours.
At the end of said period, the cells have been recovered from the culture medium and lysed by means of lysozym treatment and sonication.
The proteins so extracted have been analyzed to determine the presence of an enzymatic activity.
In practice the ADP-ribosylation activity of said proteins has been tested operating according to the method described by Manning et al. (1984) (J. Biol. Chem. 259, 749-756).
The results obtained, listed in table I of the following example 2, show that S1 sequences following the aminoacid in position 179, are not necessary for the ADP-ribosylation activity, unlike the first ten aminoacids.
The enzymatically active region of the S1 subunit, therefore, is comprised between the 1 and 180 aminoacids.
According to that, in accordance with the present invention, the identification of the active sites present in said region has been performed and at least one of said sites has been modified.
In practice, the gene coding for S1 has been isolated from the PTE 255 plasmid, the construction of which is reported in the copending Italian patent application No. 19208-A/86, by means of digestion with the restriction enzymes EcoRI and HindIII.
The 600 base pairs DNA fragment, comprising the gene coding for S1, has been separated from the digestion mixture by means of gel electrophoresis and, after electro-elution, has been modified by direct mutagenesis which allows to introduce in vitro mutations in determined sites of a DNA molecule and to test in vitro or in vivo the effect of said mutation.
By this method the substitution of the desired base it is made possible operating in one of the following ways:
by incorporating base analogues in DNA sites;
by incorporating in a wrong way nucleotides;
by introducing the mutation during the synthesis in vitro of oligonucleotides with definite sequences;
by using specific chemical mutagen agents, such as sodium bisulfite, which react with the DNA bases.
According to the present invention, the gene coding for S1 has been modified by using synthetic oligo-nucleotides with definite sequences operating according to the method described by Zoller M. J. et al. (DNA 3:479-488, (1984)).
In practice, the 600 bp DNA fragment has been cloned in a vector which allows the isolation of the single helix clone fragment of the DNA.
To this end, suitable vectors may be selected from Bluescript SK (Stratagene S. Diego, Calif.), pEMBL (Dente et al. Nucleic Acids Research 11, 1645-1655 (1983) or M13 phages (Vierra and Messing (1982) Gene, 19, 263).
According to the present invention the commercially available Bluescript SK vector has been employed.
Said vector has been treated with suitable restriction enzymes and then linked to the 600 bp DNA fragment in ligase mixture in presence of the T4 DNA ligase enzyme.
The mixture has been then employed to transform E. coli cells and the transformants have been successively selected on a culture medium including ampicillin.
The positive clones, containing the hybrid plasmids comprising the vector and the 600 bp DNA fragment, have been suspended in a liquid medium in the presence of phages and maintained at a temperature of from 30xc2x0 C. to 40xc2x0 C. for a period of from 2 to 10 hours.
At the end of said period, the phages have been precipitated, separated from the solution by centrifugation, resuspended in a pH 7.5 buffer, extracted with water-ethyl ether saturated phenol and then extracted with ethanol and ammonium acetate in order to precipitate the single helix DNA.
Aliquots of said DNA have then been employed to modify the S1 gene by direct mutagenesis. To this end oligo-nucleotides of about 20 nucleotides have been synthesized in which the bases which code for one or more aminoacids present in determined sites of the 1-180 S1 region have been substituted with others which code for a different aminoacid. In particular oligonucleotides have been synthesized which allow to prepare the following mutants of the gene coding for S1:
41: Tyrosine 8 and arginine 9 are substituted with Aspartic and Glycine respectively using the primer GTCATAGCCGTCTACGGT.
The corresponding gene has been modified in this way:
620-CGCCACCGTATACCGCTATGACTCCCGCCCG-650
620-CGCCACCGTAGACGGCTATGACTCCCGCCCG-650
22: Phenylalanine 50 and threonine 53 are substituted with glutamic acid and isoleucine respectively using the primer TGGAGACGTCAGCGCTGT.
The corresponding gene has been modified in this way:
The sequence 750-AGCGCTTTCGTCTCCACCAGC-770 has been changed into 750-AGCGCTGACGTCTCCATCAGC-770.
25: Glycine 99 has been substituted with glutamic acid using the primer CTGGCGGCTTCGTAGAAA.
The corresponding gene has been so modified:
the sequence 910-TACGGCGCCGC-920 has been changed into 910-TACGAAGCCGC-920.
17: Aspartic acid 109 has been substituted with glycine using the primer CTGGTAGGTGTCCAGCGCGCC.
The corresponding gene has been so modified:
the sequence 930-GTCGACACTTA-940 has been changed into 930-GTCGGCACTTA-940.
27: Glycine 121 has been substituted with glutamic acid using the primer GCCAGCGCTTCGGCGAGG.
The corresponding gene has been so modified:
the sequence 956-GCCGGCGCGCT-966 has been changed into 956-GCCGAAGCGCT-966.
16: Alanine in 124 position has been substituted with aspartic acid using the primer GCCATAAGTGCCGACGTATTC.
The corresponding gene has been so modified:
the sequence 976-TGGCCACCTAC-984 has been changed into 976-TGGACACCTAC-986.
1716: contains the combined 16 and 17 mutations.
28: Glutamic acid 129 has been substituted in glycine using the primer GCCAGATACCCGCTCTGG.
The corresponding gene has been so modified:
the sequence 990-AGCGAATATCT-1000 has been changed into 990-AGCGGGTATCT-1000.
29: Arginine 135 has been substituted with glutamic acid using the primer GCGGAATGTCCCGGTGTG.
The corresponding gene has been so modified:
the sequence 1010-GCGCATTCCGC-1020 has been changed into 1010-GGACATTCCGC-1020.
31: Threonine 159 has been substituted with lysine using the primer TACTCCGTTTTCGTGGTC.
The corresponding gene has been so modified:
1070-GCATCACCGGCGAGACCACGACCACGGAGTA-1090 has been changed into 1070-GCATCACCGGCGAGACCACGAAAACGGAGTA-1090.
26: Tyrosine 111 is substituted with glycine.
Furthermore, owing to a partial duplication of a primer fragment, the insertion of the Asp Thr Gly Gly aminoacids occurred in position 113 using the primer CGCCACCAGTGTCGACGTATTCGA. The corresponding gene has been so modified:
930-GTCGACACTTATGGCGACAAT-950 930-GTCGACACTGGTGGCGACACTGGTGGCGACAAT-950.
Said oligonucleotides have been used as primers for DNA polymerase which transcribes all the nucleotidic sequence of the vector incorporating the mutations present in the primer.
The vectors containing the S1 gene with the desired modification have been isolated by the hybridization technique using as probe the primer itself.
The exact nucleotide sequence of the modified gene has been then confirmed by the technique of Sanger F. et al. (P.N.A.S. 74, 5463, 1977).
The vectors containing the modified genes have been then digested with the restriction enzymes EcoRI and HindIII and the DNA fragments containing the gene coding for the modified S1 have been cloned in an expression plasmid selected from those known in the art.
Said hybrid plasmids have been employed to transform a host microorganism selected among E. coli, Bacillus subtilis and yeasts.
In particular, according to the present invention, the plasmid PEx34(Center for Molecular Biology, Heidelberg, Federal Republic of Germany) and the microorganism E. coli K12-xcex94Hl-xcex94trp (Remant, E. et"" al. Gene, 15, 81-93, 1981) have been employed.
The transformed microorganisms have been then cultivated in a liquid culture medium in the presence of carbonium, nitrogen and mineral salt sources, at a temperature comprised between 30xc2x0 C. and 45xc2x0 C. for a period of from 20 minutes to 5 hours.
At the end of the period the cells have been recovered from the culture medium by centrifuging and lysed by means of generally known techniques.
The cellular lysates containing the proteins have been then analyzed to determine the enzymatic activity thereof.
The results, reported in the following example 3, show that a good reduction (5-80%) of the ADP-ribosylation activity and therefore of toxicity has been obtained by substituting in the S1 sequence the aminoacids in 109 (17) and 124 (16) positions, either separately or in combination, and the aminoacid in 121 position (27).
A complete loss of the S1 subunit enzymatic activity has been observed by substituting the aminoacids in the positions 8 and 9 (41), 50 and 53 (22) and 129 (28).
Furthermore, said subunits are able to induce in vivo specific antibodies and to react (subunit 28) with anti-PT protective monoclonal antibodies.
Polypeptides containing said modified subunits, therefore, are suitable for the preparation of an antipertussis vaccine.
Preferred are the polypeptides containing in addition to the modified S1 subunit at least one of the S2, S3, S4 and S5 PT subunits.
Particularly preferred are the polypeptides having said S2, S3, S4 and S5 subunits with the same arrangement and configuration presented by the natural pertussis toxin.
Said preferred polypeptides may be prepared modifying the gene coding for S1 contained in the PT operon and constructing plasmids, comprising the whole operon with the modified S1 gene or regions thereof, which essentially code for a polypeptide containing the modified S1 subunit and one or more of the S2, S3, S4 and S5 subunits.
According to the present invention,the plasmids PTE 255-22, PTE 255-28 and PTE 255-41, containing the gene which codes for the S1 modified subunits 22, 28 and 41 respectively, have been deposited as E. coli (PTE 255-22), E. coli (PTE 255-28) and E. coli (PTE 255-41) at the American Type Culture Center as ATCC 67542, ATCC 67543 and ATCC 67544.